Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N2021/6417—Spectrofluorimetric devices
  • G01N2021/6419—Excitation at two or more wavelengths
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N2021/6417—Spectrofluorimetric devices
  • G01N2021/6421—Measuring at two or more wavelengths
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N2021/6491—Measuring fluorescence and transmission; Correcting inner filter effect
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
  • G01N21/274—Calibration, base line adjustment, drift correction
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2201/00—Features of devices classified in G01N21/00
  • G01N2201/02—Mechanical
  • G01N2201/022—Casings
  • G01N2201/0221—Portable; cableless; compact; hand-held
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2201/00—Features of devices classified in G01N21/00
  • G01N2201/06—Illumination; Optics
  • G01N2201/062—LED's
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2201/00—Features of devices classified in G01N21/00
  • G01N2201/06—Illumination; Optics
  • G01N2201/062—LED's
  • G01N2201/0627—Use of several LED's for spectral resolution

Definitions

• the present invention relates to a method for characterizing pigmented biological ur. It also relates to a system using this method.
• the invention relates to a method and a sy to characterize a tissue of a biological entity, such as a comprising a first chromophoric compound with little or no fluorescence as an anthocyanin or a flavonol, and a second fluorescent chromium compound. , such as chlorophyll.
• the characterization aims to determine the content of the first compound of the biological tissue.
• a characteristic of a biological tissue is of great interest. For example, in the winemaking domain, the anthocyanin content of grape berries represents information on their maturity and the quality of the wine that will be made from these berries.
• the flavonols in the skin of fruits and vegetables is an indicator of their nutritional value.
• An object of the invention is to provide a novel method and system for characterizing a biological tissue beyond saturation non-destructively, non-invasively.
• Another object of the invention is to propose a new method and a system for characterizing a biological tissue beyond saturation and usable in situ.
• the invention thus proposes a method for determining, the content of a chromophoric and non-fluorescent compound, said first compound, a biological tissue of a biological entity, the biological tissue further comprising a chromophore and fluorescent compound, said second compound this method comprising at least one iteration of the following operations:
• a first optical radiation called a measurement
• a second optical radiation referred to as a reference
• the method according to the invention is characterized in that it furthermore comprises at least one compensation of a measurement saturation due to an excessive absorption of the measurement radiation by the first compound, due to a too high content of the first compound the compensation comprising a choice, for the measurement radiation, of a wavelength corresponding to a lower absorption in the absorption spectrum of the first compound.
• the method according to the invention therefore makes it possible to carry out the characterization of a biological tissue of a biological entity, beyond a saturation due to a too high content of the first compound, that is to say, to a too much high absorption of the measurement radiation by the first compound, by shifting the wavelength of the measuring radiation to a wavelength of lower absorption in the absorption spectrum of the first compound.
• the method according to the invention is non-destructive, non-invasive and can be implemented in situ.
• the method according to the invention may further comprise an emission, by the emission means, towards the tissue, of a third optical radiation, said additional, chosen so as to induce a fluorescence radiation of the second compound.
• the method according to the invention comprises a measurement of the fluorescence radiation induced by the additional radiation. This measurement is then used to determine whether or not the reference radiation is affected by the first compound. Indeed, as a function of the ratio of the fluorescence radiation induced by the two radiations, reference and additional, and the variation of this ratio, it is possible to determine whether or not the reference radiation is affected by the first compound. When it is detected that the reference radiation is affected by the first compound, it is necessary to perform a saturation compensation, for example by inverting the measured ratio.
• the wavelength chosen for the measurement radiation after saturation corresponds to a length of wave located around the wavelength of the reference radiation before saturation.
• a new wavelength is chosen for the measurement radiation.
• the new wavelength chosen for the measurement radiation may be the wavelength of the reference radiation before saturation.
• the wavelength chosen for the reference radiation after saturation may correspond to a wavelength that is not not or little affected by the content of the first compound. More particularly, the wavelength of the reference radiation after saturation may correspond to the wavelength of the additional radiation.
• the wavelength chosen for the measurement radiation when the reference wavelength is not affected by the content of the first tissue compound, corresponds to a lower absorption by the first compound that a wavelength, called limit, for which a potential saturation would occur for a maximum expected level of the first compound in the biological tissue. In a series of measurements, the maximum potential content of the first compound can be determined.
• a maximum absorption that can potentially be obtained can be calculated.
• a wavelength, called limit, corresponding to saturation for maximum absorption can be determined for the measurement radiation.
• the limiting wavelength can be determined according to an absorption peak. In this case, the measurement wavelength is chosen with respect to the absorption peak.
• the method according to the invention may then comprise a determination of the offset with respect to the absorption peak so as to avoid saturation or obtain values of the content of the first compound evolving more linearly.
• the compensation of the saturation can be performed when the content of the tissue in the first compound causes a measurement saturation, said compensation being repeated at each saturation.
• the wavelength of the measurement radiation can be shifted to a second wavelength corresponding to a lower absorption than the first wavelength so that this second wavelength does not cause measurement saturation.
• a second series of measurement can be conducted, until a new saturation.
• the wavelength of the measurement radiation can then be shifted to a third wavelength corresponding to a lower absorption than the second wavelength, and so on.
• the difference in absorption can be taken into account when calculating the content of the first compound in the tissue. This difference in absorption can be obtained by studying the absorption spectrum of the first compound providing calibration values for the various wavelengths chosen.
• the method according to the invention may comprise a modification of the wavelength of the measurement radiation and / or of the reference radiation as a function of a physicochemical characteristic having an influence on the absorption spectrum of the first compound and or on the fluorescence spectrum of the second compound.
• the wavelength of the measurement radiation and / or the reference radiation may also be modified as a function of an appearance in the biological tissue of a new compound, for example by a covalent change, modifying the absorption spectrum of the first compound or the fluorescence spectrum of the second compound.
• the measurement radiation (or the reference radiation) can be emitted at a predetermined intensity and the reference radiation (or measurement radiation) at a variable intensity so that the fluorescence radiation induced by each of said radiations reference and measurement are equal in intensity.
• the method according to the invention may advantageously comprise a synchronization by phase modulation of the radiation emitted by the transmitting means.
• Each of the measurement and reference radiations can be transmitted in the form of pulses.
• the determination of the content of the first compound may comprise a calculation of the ratio (fluorescence induced by the reference radiation) divided by (fluorescence induced by the measurement radiation).
• the method according to the invention may advantageously comprise a determination of the evolution, in time, of the content of the first compound of the biological tissue.
• This evolution can be determined according to a unit of time that can be the day. It may, for example be a curve, showing the evolution of the content of the compound according to the unit of time chosen.
• the biological tissue is the skin of a grape berry
• the first compound is an anthocyanin
• the second compound is chlorophyll
• the wavelength of the reference radiation before saturation is 650 nm; the wavelength of the additional radiation is 450 nm;
• the wavelength of the measurement radiation before saturation is 530 nm; the wavelength of the reference radiation before saturation is 650 nm;
• the wavelength of the measurement radiation after saturation is 650 nm
• FRFred corresponds to the fluorescence radiation of the chlorophyll induced by radiation emitted at 650 nm, that is to say the reference radiation before saturation and the measurement radiation after saturation;
• FRFgreen corresponds to the fluorescence radiation of chlorophyll induced by radiation emitted at 530 nm, that is to say the measuring radiation before saturation
• FRFblue corresponds to the fluorescence radiation of the chlorophyll induced by radiation emitted at 450 nm, that is to say the additional radiation before saturation and the reference radiation after saturation
• Expressions 3 and 4 correspond to at least one measurement carried out before the appearance of anthocyanins in the grape skin. It serves as a reference for measurements made after the appearance of anthocyanins;
• the biological tissue is the skin of a grape berry
• the first compound is a flavonol
• the second compound is chlorophyll:
• the wavelength of the measurement radiation before saturation is around 375 nm
• the wavelength of the reference radiation before saturation is around 650 nm
• FIG. 1 is a schematic representation of a first embodiment of the system according to the invention
• FIG. 2 is a schematic representation of a second embodiment of the system according to the invention.
• FIG. 3 is a schematic representation of the measuring principle in the first embodiment of the system according to the invention.
• FIG. 5 is a schematic representation of a system according to the second embodiment.
• FIG. 11 is a representation of the results obtained when measuring the anthocyanin content of the skin of a grape berry according to the second aspect of the process according to the invention.
• FIG. 12 is a representation of the results obtained when measuring the anthocyanin content of the skin of a grape berry according to a combination of the first and second aspects of the process according to the invention
• FIG. 13 is a representation of an example of variation of the absorption spectrum of anthocyanins as a function of the pH
• FIGS. 1 and 2 are diagrammatic representations of two embodiments of a system according to the invention. In these two embodiments, the system is in the form of two parts: a transmitting part 10 and a receiving part 20. In the first embodiment shown in FIG.
• the emitter 10 and the receiving part 20 are disposed of and other a sample of a plant tissue 30, while in the second embodiment shown in Figure 2, the emitter 10 and receiver 20 are disposed on the same side of a biological tissue sample 30.
• the emitting part 10 comprises three radiation sources 11, 12 and 13 illuminating the front face of a sample of biological tissue 30. Each of the sources 11 to 13 is associated with a pulsed supply 14 to 16, as shown in FIGS. 1 and 2, and controlled by a synchronization signal S 5 . Sources 11 to 13 are provided to induce the fluorescence of chlorophyll.
• the receiving part 20 of the system further comprises a block 22 comprising control and calculation means making it possible to one part of synchronizing the power supplies 14 to 16 via a synchronization signal S s and determine the content of the first compound of the tissue sample 30 as a function of the measurement signal S m provided by the detector 21.
• Figures 3 and 4 are schematic representations of the measuring principle and the optical paths of the different radiations, respectively in the first and the second embodiment.
• the sources 11 to 13 are preferably light-emitting diodes (LEDs) and illuminate the same face 31 of the sample 30.
• the sources 11 to 13 emit three radiations, respectively 111, 121 and 131, intended to induce the fluorescence of chlorophyll 33 and to induce three fluorescent radiations, respectively 112, 122 and 132.
• the receiving part 20 of the system located on the opposite side of the sheet or bay with respect to the emitting part 10, it is the fluorescence radiation emitted by the chlorophyll towards the opposite side to the sources 11 to 13 which will be detected by the detector. 21.
• the detector 21 is provided to detect the fluorescence emitted towards the rear face 35 of the sample to measure the polyphenol content 34.
• FIG. 4 represents the fluorescent radiation 112, 122 and 132 induced by the radiation 111, 121 and 131, and emitted by the chlorophyll towards the sources 11, 12 and 13.
• the receiving part 20 of the system lying on the same side of the sheet or bay as the emitting part 10, it is the radiation emitted by the chlorophyll towards the sources 11 to 13 which will be detected by the detector 21.
• the detector 21 is provided to detect the fluorescence emitted towards the front face 31 of the sheet to measure the polyphenol content 34.
• the wavelengths of each of the sources 11 to 13 are chosen according to the absorption bands of the compounds to be measured, their technical characteristics such as the spectral purity or their power, their commercial availability and their cost, and the commercial availability and cost of the F2 or filters associated with the detector 21.
• the first and second embodiments are substantially equivalent because the fluorescence is isotropic.
• the second embodiment is the preferred embodiment and makes it possible to use the system according to the invention in situ and at a distance from the biological tissue to be characterized.
• the system according to the invention can be used directly on the biological entity in a non-destructive and non-intrusive manner, that is to say without having to carry out sampling of a sample of biological tissue.
• FIGS. 5 to 7 show representations according to different views of an apparatus 50 made according to the second embodiment of the system according to the invention, this second embodiment being the preferred embodiment of the system according to the invention.
• FIG. 5 gives a representation in a sectional view
• the apparatus 50 makes it possible to measure directly on a grape cluster 60 comprising a plurality of bays 70.
• realization which we will call MULTIPLEX®, is based on a portable housing 510 and powered by a remote battery 5121 or integrated in the handle, having a measurement face 514 and a user interface comprising a screen 5152 and organs of controls such as buttons or buttons 5101 and 5102.
• This housing can be held by a portion forming a handle 512 containing the removable battery 5121 or the connector of the remote portable battery.
• This housing 510 also comprises a cylindrical portion 513 extending towards the opposite side to the interface and carrying the measuring face at its end.
• the measurement face 514 is surrounded by a more or less opaque and possibly removable cover 5130, which makes it possible to reduce the interference of the ambient light and to give a reference as to the optimum measurement distance with respect to the measuring face 514.
• This measuring face 514 comprises a set 540 of detectors covering the fluorescence wavelengths to be measured.
• this set 540 comprises three detectors 541, 542 and 543 adjacent to each other and grouped together in an equilateral triangle in the center of the measuring face 514. These three detectors are oriented in directions parallel to one another around the other. a detection axis 5140, or very slightly convergent around this detection axis 5140.
• Each of these detectors 541, 542 and 543 comprises a detection element, here a silicon photodiode 5420 of approximately 2cm ⁇ 2cm, and detects the light in a determined wavelength band, respectively blue-green, red and far-red.
• This detection band is obtained by a colored or high-pass filter and an interference filter. The combination of these two types of filters allows a better filtration which may be necessary, in particular, to prevent the detectors from receiving radiation emitted by the excitation sources.
• the detectors receive directly the fluorescence to be measured, without using collection optics, convergent or collimated.
• Each detector requires only one detection element, the photodiode 5420 (FIG. 7), chosen sufficiently large to obtain a good sensitivity that makes it possible to dispense with collection optics.
• This detection element thus receives a radiation 549 coming from the whole of the target zone 591 illuminated by the excitation emitters.
• the excitation emitters are oriented so as to obtain uniform illumination of the target zone 591, even when it is a heterogeneous and / or three-dimensional object.
• the emitted beams are not collimated and have a certain opening or divergence makes it possible to limit the constraints on the measurement distance. Indeed, as the target, here a bunch of grapes 60, is inside the beams 529 and 539 of the emitters, it will be illuminated homogeneously on its different faces directed towards the set 540 of detectors. The fluorescence 549 emitted towards the detectors will thus be sufficiently stable and homogeneous to provide accurate measurements of the measured area 591.
• the measurements using the UV emitters are made at a distance of about 15 cm
• the measurements using only the emitters in visible light can be carried out at greater distances, or even to around Im, by example for a measurement of anthocyanins.
• the beams of the emitters may be oriented so as to be parallel to the detection axis, for example for measurement applications at a large distance.
• FIG. 7 illustrates more particularly the structure of the device in this embodiment of the invention.
• the cylindrical portion 513 of the housing encloses a substantially annular electronic card 5131, comprising the power supply circuits of the excitation sources, the excitation pulse management circuit and a circuit producing a current generator.
• the detectors, and the electronics that go with them, are grouped in a cylindrical detection module 5400, placed on the measuring face 514 at the center of the circle formed by the excitation emitters and extending in the direction of the target at analyze.
• the outer face of this cylinder carries the three detectors 541, 542 and 543.
• This arrangement makes it possible to place the detectors substantially at the same level as the ends of the emitters so that they have a wide reception field and thus makes it possible to approach the target.
• the detection module 5400 comprises three small substantially analog electronic cards 5141, 5142, 5143, substantially circular, stacked along its longitudinal axis, fixed and spaced by columns 5144.
• the first small electronic card 5141 located on the side of the outer face of the detection module 5400, carries the detection elements, here silicon photodiodes.
• the silicon photodiode 5420 receives the light to be detected through a colored or high-pass filter 5421 and an interference filter 5422 removably fixed by a locking nut 5423 in a cylindrical opening of 25.4 mm, thus able to receive standard filters of one inch or 25mm.
• the detection module 5400 constitutes a compact assembly that can be removed from the housing 510, for example for maintenance or to be replaced by a camera module or a module including one or more optical guides.
• management and processing means are arranged for:
• control for example by the same control signal, the processing of the fluorescence detection signal by the sample-and-hold devices; and - provide an analog measurement of the fluorescence measurement at the datalogger.
• synchronous detection using phase modulation between excitation and detection is provided.
• the management and processing means are then arranged to:
• FIG. 8 gives the absorption spectrum 82 of malvidin glycoside, which is the major anthocyanin of grapes, in acidified 50% methanol, in unit of extinction coefficients, the emission spectrum 83 of chlorophyll and the transmission spectrum 84 of the F2 filter which is a Schott RG9 filter.
• the source 11 is a source emitting radiation 111 in the green (GREEN), the wavelength of which lies in an absorption band of the anthocyanins and which induces the fluorescence of the chlorophyll 33 and causes the emission of a Fluorescence radiation 112.
• the wavelength of this source 111 may for example be around 530 nm.
• FIG. 8 gives the spectrum 86 of the radiation 11.
• An example of a GREEN source is the NS530L diode from Roithner Lasertechnik.
• the source 12 emits red 121 radiation (RED), in which the anthocyanins do not absorb or little, which induces the fluorescence of the chlorophyll 33 and causes the emission of fluorescence radiation 122, and which serves as a reference for the measurement of the anthocyanin content.
• RED red 121 radiation
• the wavelength of this source is preferably 650 nm.
• Figure 8 gives the spectrum 85 of the radiation 121.
• the source 13 emits radiation 131 in the blue (BLUE), where the anthocyanins absorb little, which induces the fluorescence of the chlorophyll 33 and causes the emission of a fluorescence radiation 132, which serves as additional radiation for the measurement of the anthocyanin content.
• the wavelength of this source is preferably 450 nm.
• Figure 8 gives the spectrum 87 of the radiation 12.
• the sources 11, 12 and 13 successively illuminate the tissue 30 by emission respectively of radiation 111, 121 and 131.
• the radiation 111 is absorbed in variable quantity by the epidermis of the skin according to its anthocyanin content 34, while the red radiation
• the measurement of the ratio of the fluorescence emissions excited by the source 11 and the source 12 allows to determine the anthocyanin content of the skin of a grape berry.
• the measurement of the ratio of the fluorescence emissions excited by the source 12 and the source 13 makes it possible to determine whether the reference radiation 12 is affected by the anthocyanins whose content increases over time. When the anthocyanin content of a grape berry increases, the radiation 111 is too absorbed and begins to be saturated. In addition, anthocyanins also begin to affect the reference radiation.
• the radiation 121 which was the reference radiation before saturation
• the radiation 131 which was the additional radiation before saturation
• the radiation 111 is no longer used in the measurement.
• the anthocyanin content is then calculated according to the following expression:
• logFER_ANTH_RED log (FRFbI ue / FRFred)
• logFER_CHL G log (FRFred / FRFgreen)
• FIG. 9 gives a graph showing the evolution of the content measured in anthocyanins, in the skin of a grape berry, determined according to the first aspect of the invention.
• the axis 91 designates the moment from which the compensation of the saturation is carried out.
• Curve 92 shows the results obtained with compensation of the saturation according to the invention, that is to say considering the reference radiation before saturation as measurement radiation after saturation and the additional radiation before saturation as reference radiation. after saturation.
• Curve 93 shows the results obtained without compensation for saturation.
• a decreasing curve 93 is obtained after saturation while the anthocyanin content continues to increase.
• a growing curve 92 is obtained, witnessing the increase in the anthocyanin content of the skin of a grape berry.
• the wavelength of the radiation 121 remains unchanged at 650 nm.
• the wavelength of the radiation 111 is chosen at 590 nm. This wavelength corresponds to a lower absorption of anthocyanins than the wavelength 530nm chosen according to the first aspect of the invention and which corresponds to the absorption peak of anthocyanins.
• the absorption spectrum 82 of the malvidin glycoside which is the anthocyanin whose content is to be determined, in acidified 50% methanol, in unit of extinction coefficients, the emission spectrum 83 of the chlorophyll and the transmission spectrum 84 of the filter F2, as well as the spectrum 85 of the radiation 121 and the spectrum 88 of the radiation 111 used in this second aspect of the invention.
• the measurement principle in this second aspect of the invention is substantially similar to the measurement principle in the first aspect of the invention.
• the sources 11 and 12 successively illuminate the tissue 30 by emission respectively of the radiation 111 and 121.
• the radiation 111 is absorbed in a variable quantity by the epidermis of the skin according to its content in anthocyanins 34, whereas the red radiation 121 does not. is not.
• These radiations 111 and 121 induce the fluorescence of chlorophyll 33 which then emits respectively fluorescence radiation 112 and 122 which are measured.
• the first and second aspects of the invention may be combined.
• the additional radiation 131 described above can be used to determine whether the reference radiation 121 is affected or not by anthocyanins.
• concentration or the content of anthocyanins is such that a saturation occurs even with a radiation 111 at a wavelength shifted with respect to the absorption peak, for example 590 nm
• the radiation 121 which was the reference radiation before saturation
• radiation 131 which was the additional radiation before saturation
• Figure 12 shows the results obtained by combining the first and second aspects of the invention.
• Curve 1201 corresponds to the results of measurements made with:
• the curve 1202 corresponds to the results obtained with: a radiation 111 of 590 nm;
• the axis 1203 designates the moment from which the saturation compensation is performed according to the first aspect of the invention.
• the wavelengths of the radiation 111, 121 and 131 may be modified as a function of the pH change in the biological tissue that is to be characterized.

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• Health & Medical Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Physics & Mathematics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
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• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
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• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

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